I encountered the following error during the dada2 denoise-paired step after removing primers using cutadapt (see below for the command and error). The amplicon has an average length around 183 bp. I'm using qiime2-amplicon-2024.5 on a mac M3 max computer. I've run this exact code for many other libraries, all prepped exactly the same using the same assay, but have never encountered this error. I did notice that the R1 & R2 fastq.gz files for one sample contain no readable data. Could this explain the error? I'm rerunning now after removing that sample to see if it works, but I was hoping someone here may have some insights. Thanks!
(qiime2-amplicon-2024.5) rdceradk@CERADK-MN-BB888 Trinity_Orchid_inverts_09Dec2024 % qiime dada2 denoise-paired
--i-demultiplexed-seqs demux-paired-end-trimmed.qza
--p-trunc-len-f 125
--p-trunc-len-r 125
--p-trim-left-f 0
--p-trim-left-r 0
--p-n-threads 15
--p-n-reads-learn 1000000
--o-representative-sequences rep-seqs-dada.qza
--o-table table-dada.qza
--o-denoising-stats stats-dada.qza
--verbose
Running external command line application(s). This may print messages to stdout and/or stderr.
The command(s) being run are below. These commands cannot be manually re-run as they will depend on temporary files that no longer exist.
Command: run_dada.R --input_directory /var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/forward --input_directory_reverse /var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/reverse --output_path /var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/output.tsv.biom --output_track /var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/track.tsv --filtered_directory /var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/filt_f --filtered_directory_reverse /var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/filt_r --truncation_length 125 --truncation_length_reverse 125 --trim_left 0 --trim_left_reverse 0 --max_expected_errors 2.0 --max_expected_errors_reverse 2.0 --truncation_quality_score 2 --min_overlap 12 --pooling_method independent --chimera_method consensus --min_parental_fold 1.0 --allow_one_off False --num_threads 15 --learn_min_reads 1000000
R version 4.3.3 (2024-02-29)
Loading required package: Rcpp
DADA2: 1.30.0 / Rcpp: 1.0.12 / RcppParallel: 5.1.6
2) Filtering The filter removed all reads: /var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/filt_f/4March2024-76_L001_R1_001.fastq.gz and /var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/filt_r/4March2024-76_L001_R2_001.fastq.gz not written.
Some input samples had no reads pass the filter.
........................................................................................................................................................................................................................x............................................................
3) Learning Error Rates
312203250 total bases in 2497626 reads from 3 samples will be used for learning the error rates.
312203250 total bases in 2497626 reads from 3 samples will be used for learning the error rates.
3) Denoise samples ................................................................Error in dada(drpF, err = err, multithread = multithread, verbose = FALSE) :
NAs in derep$quals matrix. Check that all input sequences had valid associated qualities assigned.
In addition: Warning message:
In derepQuals[sqnms, ] + out$cum_quals[sqnms, ] :
NAs produced by integer overflow
2: stop("NAs in derep$quals matrix. Check that all input sequences had valid associated qualities assigned.")
1: dada(drpF, err = err, multithread = multithread, verbose = FALSE)
Traceback (most recent call last):
File "/opt/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2_dada2/_denoise.py", line 350, in denoise_paired
run_commands([cmd])
File "/opt/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2_dada2/_denoise.py", line 37, in run_commands
subprocess.run(cmd, check=True)
File "/opt/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/subprocess.py", line 528, in run
raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['run_dada.R', '--input_directory', '/var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/forward', '--input_directory_reverse', '/var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/reverse', '--output_path', '/var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/output.tsv.biom', '--output_track', '/var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/track.tsv', '--filtered_directory', '/var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/filt_f', '--filtered_directory_reverse', '/var/folders/tf/9kbmk08x4y19x3rhtkqd_cjm0000gr/T/tmputofb3vt/filt_r', '--truncation_length', '125', '--truncation_length_reverse', '125', '--trim_left', '0', '--trim_left_reverse', '0', '--max_expected_errors', '2.0', '--max_expected_errors_reverse', '2.0', '--truncation_quality_score', '2', '--min_overlap', '12', '--pooling_method', 'independent', '--chimera_method', 'consensus', '--min_parental_fold', '1.0', '--allow_one_off', 'False', '--num_threads', '15', '--learn_min_reads', '1000000']' returned non-zero exit status 1.
During handling of the above exception, another exception occurred:
Traceback (most recent call last):
File "/opt/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2cli/commands.py", line 520, in call
results = self._execute_action(
File "/opt/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2cli/commands.py", line 581, in _execute_action
results = action(**arguments)
File "", line 2, in denoise_paired
File "/opt/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/qiime2/sdk/action.py", line 342, in bound_callable
outputs = self.callable_executor(
File "/opt/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/qiime2/sdk/action.py", line 576, in callable_executor
output_views = self._callable(**view_args)
File "/opt/miniconda3/envs/qiime2-amplicon-2024.5/lib/python3.9/site-packages/q2_dada2/_denoise.py", line 363, in denoise_paired
raise Exception("An error was encountered while running DADA2"
Exception: An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.
Plugin error from dada2:
An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.
See above for debug info.